0:16Skip to 0 minutes and 16 secondsimmature stages: the eggs, larvae, and pupae, of Aedes aegypti mosquitoes are found in a variety of water holding containers in or around houses. You have also learned that entomological surveys of Aedes aegypti are important to determine the effect of control measures on mosquito populations and/or to study epidemiology of vector borne to disease. Historically, surveillance of Aedes mosquitoes has relied more heavily on estimating the population densities of the immature stages rather than the adults. This is because it was comparatively harder to collect Aedes aegypti adults than immatures due to their activity during the day, and their tendency to rest and feed outdoors.

1:02Skip to 1 minute and 2 secondsAs you've heard in the previous step, trapping and collection methods for capturing adult Aedes aegypti mosquitoes are becoming more widely available and they provide better data for quantifying transmission risks than larval counting. Nevertheless, obtaining densities of immature stages still has operational value because they're used to study the local vector ecology and to measure the impact of vector control interventions aimed to reduce container breeding. In theory it should be relatively easy to count larvae, but as you can see from this slide, showing photos taken in Puerto Rico, the variety and the limited access of many natural breeding sites poses challenges.

1:46Skip to 1 minute and 46 secondsYou can find immature stages of Aedes in small, temporary containers such as rain filled plants, discard items, and tyres, as well as more permanent containers such as water meters and oil drums. Therefore, obtaining reliable estimates requires searching.

2:05Skip to 2 minutes and 5 secondsSo what does a breeding site with Aedes larvae look like and how do I count the larvae? You need to examine all containers with water present. If there is larvae in the water the first thing that you will notice is movement, so inspect these containers more carefully. The larvae will move up to the surface of the water to breathe and will then move down to the bottom to feed. You can see Aedes larvae at the surface of the water on the photograph on the left. The round structure at one end of the body is the head and the pointed structure at the other end is the siphon. The siphon allows them to penetrate the surface of water and to respire.

2:47Skip to 2 minutes and 47 secondsThere is no standard equipment for sampling the larvae but for large containers a dipper, as shown in the photograph on the right, or a net may be used but for small containers the entire contents are emptied on to a tray and the immature stages are picked out using a pipette. If necessary, the samples can be stored in vials labelled with details relating to their data collection sampling site and then taken back to the laboratory for species identification. You will find out more about how to prepare your samples later in the course.

3:24Skip to 3 minutes and 24 secondsLarval surveys involve identifying all or most of the immature mosquitoes found in every container, or a representative sample of containers in the target area. The traditional indices used to study the impact of

3:37Skip to 3 minutes and 37 secondsvector interventions are: the house index, which is the percentage of houses including the surrounding compound that have larvae of Aedes aegypti in at least some of the containers. The container index is the percentage of water holding containers examined that contain larvae of Aedes aegypti.

3:59Skip to 3 minutes and 59 secondsThe Breteau index is the total number of containers with larvae of Aedes aegypti in 100 houses being sampled. The larval density index, which is the mean number of Aedes aegypti per house, is used less often because it is laborious as it requires counting all of the larvae present in each container.

4:24Skip to 4 minutes and 24 secondsThe WHO produced a table to compare larval densities throughout the world in order to identify population sizes of Aedes aegypti which represent a threat of urban transmission of yellow fever. For dengue, a house index of greater than 10% or Breteau index of greater than 50 is considered a high risk of transmission, whereas a house index of less than 1% or Breteau index of less than 5 is considered low risk. No estimates have been proposed for Zika. Note that these figures need to be considered very cautiously. The limitations of these indices are that productivity of containers varies greatly over time and larval density cannot be used to predict adult numbers, therefore traditional larval indices have limited use for assessing transmission risk.

5:21Skip to 5 minutes and 21 secondsHowever, larval indices are useful for identifying whether certain containers pose more risk of harbouring larvae than others. And this means that operational programmes focusing on containers, either by treating them with insecticides or removing them from an environment can be more targeted.

5:43Skip to 5 minutes and 43 secondsPupal surveys for the number of pupae per person is calculated, provide a better proxy than larvae for use in risk thresholds. Pupal surveys normally require a pair of collectors sampling all of the water filled containers in and around 100 or more houses, having previously obtained informed consent from the householders. The water is sieved and the pupae counted. If there are several container breeding species in the area it cannot be assumed that all pupae present are Aedes aegypti, therefore the larvae need to be placed in vials and returned to the laboratory where they can emerge as adults for species identification. So why count pupae?

6:28Skip to 6 minutes and 28 secondsSo in contrast to the other life stages it is possible to determine the absolute number of Aedes aegypti pupae in most domestic environments. Secondly, because pupal mortality is low, there is a good correlation between the number of pupae and the number of adults. The counts of eggs or larvae are poor proxies because there is more mortality and the costs are prohibitive too. The third reason is that Aedes pupae can be easily removed, returned to the laboratory and identified to species when they emerge as adults. And lastly, counting pupae, like counting larvae, allows comparisons to be made regarding the productivity of different types of containers.

7:13Skip to 7 minutes and 13 secondsThe number of Aedes aegypti pupae per person is used in transmission simulation models of dengue to determine risk thresholds, and can be used to target strategies aimed at containers. For example, this table provides survey results from five locations and estimates of transmission thresholds. Under the targeted column are the proportions of containers that need to be controlled or eliminated if the programme focus only on the most productive containers in the environment. Please refer to the original source for information, Focks'paper published in 2003, because it provides more information, but it is evident that a strategic, targeted approach requires less resources than a programme that's not targeted. However, currently there is no information on how pupal indices correlate with chikungunya or Zika transmission.

8:21Skip to 8 minutes and 21 secondsLastly, we will deal with the last stage of the life cycle, which are eggs. Ovitraps exploit the tendency of Aedes aegypti and Aedes albopictus females to lay their eggs in artificial containers. Ovitraps are basically artificial oviposition sites and they come in all sorts of shapes and sizes. Generally, they are small containers, usually dark in colour, that contain water and a rough substrate such as wood, cloth, or cardboard for the female mosquitoes to lay their eggs. The typical trap shown here is a plastic, water filled container with a cardboard paddle that serves as the preferred rough substrate for the female to lay her eggs.

9:07Skip to 9 minutes and 7 secondsThe paddle is longer than the container so it can be picked up and removed without rubbing off any eggs that have been laid. Usually only presence or absence is recorded, but the number of eggs can also be counted if required. Ovitraps are normally placed in shaded areas, which are the preferred breeding sites of mosquitoes, and at selected sites where they will encounter minimal disturbance from people. Several attempts have been made to increase their sensitivity such as using different colours or types of substrates and using different chemical attractants, for example hay infusion is often used. They are usually monitored once a week before any laid eggs develop into adults. They are inexpensive, easily deployed, and are not invasive.

9:57Skip to 9 minutes and 57 secondsTypically only one ovitrap is placed per city block. However, interpreting ovitrap data requires caution. Ovitraps compete with naturally occurring larval habitats and they may not accurately reflect the abundance of gravid females. They are used to gather data on the spatial and temporal distributions of Aedes aegypti and to quantify the impact of interventions aimed at containers. They should not be used as a proxy for adult densities. In addition to monitoring and surveillance of vector populations, they can be used as lethal ovitraps when an insecticide is used with the bait for control.

Trapping and monitoring: immature stages

We’ve seen the ways in which adult mosquitoes can be trapped for surveillance, but what about the immature stages, or eggs, larvae, and pupae?

In the past we have relied more heavily on estimating the population densities of immature mosquitoes than adults in our surveillance of Aedes. In this step Dr Mary Cameron explains why that is, highlighting some of the ways in which we might survey immature stages and how we can then identify population sizes.